Thiophene-carboxamides useful as inhibitors of protein kinases

ABSTRACT

The present invention relates to compounds useful as inhibitors of protein kinase. The invention also relates to pharmaceutically acceptable compositions comprising said compounds and methods of using the compounds and compositions in the treatment of various disease, conditions, or disorders. The invention also relates to processes for preparing compounds of the inventions.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application60/802,655 filed May 23, 2006, the entire content of which isincorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on May 10, 2010, is namedVPI06125.txt and is 965 bytes in size.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to compounds useful as inhibitors ofprotein kinases. The invention also relates to pharmaceuticallyacceptable compositions comprising the compounds of the invention andmethods of using the compositions in the treatment of various disorders.The invention also relates to processes for preparing the compounds ofthe invention.

BACKGROUND OF THE INVENTION

The search for new therapeutic agents has been greatly aided in recentyears by a better understanding of the structure of enzymes and otherbiomolecules associated with diseases. One important class of enzymesthat has been the subject of intensive study is protein kinases.

Protein kinases constitute a large family of structurally relatedenzymes that are responsible for the control of a variety of signaltransduction processes within the cell (see Hardie, G and Hanks, S. TheProtein Kinase Facts Book, and II, Academic Press, San Diego, Calif.:1995). Protein kinases are thought to have evolved from a commonancestral gene due to the conservation of their structure and catalyticfunction. Almost all kinases contain a similar 250-300 amino acidcatalytic domain. The kinases may be categorized into families by thesubstrates they phosphorylate (eg protein-tyrosine,protein-serine/threonine, lipids etc). Sequence motifs have beenidentified that generally correspond to each of these kinase families(See, for example, Hanks, S. K., Hunter, T., FASEB J. 1995, 9, 576-596;Knighton et al., Science 1991, 253, 407-414; Hiles et al, Cell 1992, 70,419-429; Kunz et al, Cell 1993, 73, 585-596; Garcia-Bustos et al, EMBOJ. 1994, 13, 2352-2361).

In general, protein kinases mediate intracellular signaling by effectinga phosphoryl transfer from a nucleoside triphosphate to a proteinacceptor that is involved in a signaling pathway. These phosphorylationevents act as molecular on/off switches that can modulate or regulatethe target protein biological function. These phosphorylation events areultimately triggered in response to a variety of extracellular and otherstimuli. Examples of such stimuli include environmental and chemicalstress signals (eg shock, heat shock, ultraviolet radiation, bacterialendotoxin, and H₂O₂), cytokines (eg interleukin-1 (IL-1) and tumornecrosis factor alpha (TNF-a), and growth factors (eg granulocytemacrophage-colony stimulating factor (GM-CSF), and fibroblast growthfactor (FGF)). An extracellular stimulus may affect one or more cellularresponses related to cell growth, migration, differentiation, secretionof hormones, activation of transcription factors, muscle contraction,glucose metabolism, control of protein synthesis, survival andregulation of the cell cycle.

Many diseases are associated with abnormal cellular responses triggeredby protein kinase-mediated events as described above. These diseasesinclude, but are not limited to, cancer, autoimmune diseases,inflammatory diseases, bone diseases, metabolic diseases, neurologicaland neurodegenerative diseases, cardiovascular diseases, allergies andasthma, Alzheimer's disease and hormone related diseases. Accordingly,there has been a substantial effort in medicinal chemistry to findprotein kinase inhibitors that are effective as therapeutic agents.

The Polo-like kinases (Plk) belong to a family of serine/threoninekinases that are highly conserved across the species, ranging from yeastto man (reviewed in Lowery D M et al., Oncogene 2005, 24; 248-259). ThePlk kinases have multiple roles in cell cycle, including control ofentry into and progression through mitosis.

Plk1 is the best characterized of the Plk family members. Plk1 is widelyexpressed and is most abundant in tissues with a high mitotic index.Protein levels of Plk1 rise and peak in mitosis (Hamanaka, R et al., JBiol Chem 1995, 270, 21086-21091). The reported substrates of Plk1 areall molecules that are known to regulate entry and progression throughmitosis, and include CDC25C, cyclin B, p53, APC, BRCA2 and theproteasome. Plk1 is upregulated in multiple cancer types and theexpression levels correlate with severity of disease (Macmillan, J C etal., Ann Surg Oncol 2001, 8, 729-740). Plk1 is an oncogene and cantransform NIH-3T3 cells (Smith, M R et al., Biochem Biophys Res Commun1997, 234, 397-405). Depletion or inhibition of Plk1 by siRNA,antisense, microinjection of antibodies, or transfection of a dominantnegative construct of Plk1 into cells, reduces proliferation andviability of tumour cells in vitro (Guan, R et al., Cancer Res 2005, 65,2698-2704; Liu, X et al., Proc Natl Acad Sci USA 2003, 100, 5789-5794,Fan, Y et al., World J Gastroenterol 2005, 11, 4596-4599; Lane, H A etal., J Cell Biol 1996, 135, 1701-1713). Tumour cells that have beendepleted of Plk1 have activated spindle checkpoints and defects inspindle formation, chromosome alignment and separation and cytokinesis.Loss in viability has been reported to be the result of an induction ofapoptosis. In contrast, normal cells have been reported to maintainviability on depletion of Plk1. In vivo knock down of Plk1 by siRNA orthe use of dominant negative constructs leads to growth inhibition orregression of tumours in xenograft models.

Plk2 is mainly expressed during the G1 phase of the cell cycle and islocalized to the centrosome in interphase cells. Plk2 knockout micedevelop normally, are fertile and have normal survival rates, but arearound 20% smaller than wild type mice. Cells from knockout animalsprogress through the cell cycle more slowly than in normal mice (Ma, etal., Mol Cell Biol 2003, 23, 6936-6943). Depletion of Plk2 by siRNA ortransfection of kinase inactive mutants into cells blocks centrioleduplication. Downregulation of Plk2 also sensitizes tumour cells totaxol and promotes mitotic catastrophe, in part by suppression of thep53 response (Burns T F et al., Mol Cell Biol 2003, 23, 5556-5571).

Plk3 is expressed throughout the cell cycle and increases from G1 tomitosis. Expression is upregulated in highly proliferating ovariantumours and breast cancer and is associated with a worse prognosis(Weichert, W et al., Br J Cancer 2004, 90, 815-821; Weichert, W et al.,Virchows Arch 2005, 446, 442-450). In addition to regulation of mitosis,Plk3 is believed to be involved in Golgi fragmentation during the cellcycle and in the DNA-damage response. Inhibition of Plk3 by dominantnegative expression is reported to promote p53-independent apoptosisafter DNA damage and suppresses colony formation by tumour cells (Li, Zet al., J Biol Chem 2005, 280, 16843-16850.

Plk4 is structurally more diverse from the other Plk family members.Depletion of this kinase causes apoptosis in cancer cells (Li, J et al.,Neoplasia 2005, 7, 312-323). Plk4 knockout mice arrest at E7.5 with ahigh fraction of cells in mitosis and partly segregated chromosomes(Hudson, J W et al., Current Biology 2001, 11, 441-446).

Molecules of the protein kinase family have been implicated in tumourcell growth, proliferation and survival. Accordingly, there is a greatneed to develop compounds useful as inhibitors of protein kinases. Theevidence implicating the Plk kinases as essential for cell division isstrong. Blockade of the cell cycle is a clinically validated approach toinhibiting tumour cell proliferation and viability. It would thereforebe desirable to develop compounds that are useful as inhibitors of thePlk family of protein kinases (eg Plk1, Plk2, Plk3 and Plk4), that wouldinhibit proliferation and reduce viability of tumour cells, particularlyas there is a strong medical need to develop new treatments for cancer.

SUMMARY OF THE INVENTION

Compounds of this invention, and pharmaceutically acceptablecompositions thereof, are useful as inhibitors of protein kinases. Insome embodiments, these compounds are useful as inhibitors of PLKprotein kinases; in some embodiments, as inhibitors of PLK1 proteinkinases. These compounds have the formula I, as defined herein, or are apharmaceutically acceptable salt thereof.

These compounds and pharmaceutically acceptable compositions thereof areuseful for treating or preventing a variety of diseases, disorders orconditions, including, but not limited to, an autoimmune, inflammatory,proliferative, or hyperproliferative disease, a neurodegenerativedisease, or an immunologically-mediated disease. The compounds providedby this invention are also useful for the study of kinases in biologicaland pathological phenomena; the study of intracellular signaltransduction pathways mediated by such kinases; and the comparativeevaluation of new kinase inhibitors.

DETAILED DESCRIPTION OF THE INVENTION

This invention describes compounds of formula:

wherein

R¹ is H, C₁₋₆aliphatic, or C₃₋₆cycloaliphatic;

G is —C(R)₂— or —O—;

L is C₀₋₃aliphatic optionally substituted with 0-3 J^(L);

-   Ring A is triazolyl optionally substituted with 0-3 J^(A);-   Ring B is 5-6 membered aromatic monocyclic ring containing 0-3    heteroatoms selected from O, N, and S; Ring B is optionally    substituted with 0-5 J^(B) and optionally fused to Ring B′;-   Ring B′ is a 5-8 membered aromatic or nonaromatic monocyclic ring    containing 0-3 heteroatoms selected from O, N, and S;

Ring B′ is optionally substituted with 0-4 J^(B′);

-   each J^(A), J^(B), and J^(B′) is independently C₁₋₆haloalkyl, halo,    NO₂, CN, Q, or —Z-Q;-   Z is independently C₁₋₆aliphatic optionally interrupted with 0-3    occurrences of —NR—, —O—, —S—, —C(O)—, —C(═NR)—, —C(═NOR)—, —SO—, or    —SO₂—; each Z is optionally substituted with 0-2 J^(Z);-   Q is H; C₁₋₆ aliphatic; a 3-8-membered aromatic or nonaromatic    monocyclic ring having 0-3 heteroatoms independently selected from    O, N, and S; or an 8-12 membered aromatic or nonaromatic bicyclic    ring system having 0-5 heteroatoms independently selected from O, N,    and S; each Q is optionally substituted with 0-5 J^(Q);-   each J^(L) and J^(Z) is independently H, halo, C₁₋₆ aliphatic,    C₃₋₆cycloaliphatic, NO₂, CN, —NH₂, —NH(C₁₋₄ aliphatic), —N(C₁₋₄    aliphatic)₂, —OH, —O(C₁₋₄ aliphatic), —CO₂H, —CO₂(C₁₋₄ aliphatic),    —O(haloC₁₋₄ aliphatic), or halo(C₁₋₄ aliphatic);-   each J^(Q) is independently M or —Y-M;-   each Y is independently an unsubstituted C₁₋₆aliphatic optionally    interrupted with 0-3 occurrences of —NR—, —O—, —S—, —C(O)—, —SO—, or    —SO₂—;-   each M is independently H, C₁₋₆ aliphatic, C₃₋₆cycloaliphatic,    halo(C₁₋₄ aliphatic), —O(haloC₁₋₄ aliphatic), C₃₋₆ heterocyclyl,    halo, NO₂, CN, OH, OR′, SH, SR′, NH₂, NHR′, N(R′)₂, COH, COR′,    CONH₂, CONHR′, CONR′₂, NHCOR′, NR′COR′, NHCONH₂, NHCONHR′,    NHCON(R′)₂, SO₂NH₂, SO₂NHR′, SO₂N(R′)₂, NHSO₂R′, or NR′SO₂R′;-   R is H or unsubstituted C₁₋₆aliphatic;-   R′ is unsubstituted C₁₋₆aliphatic; or two R′ groups, together with    the atom to which they are bound, form an unsubstituted 3-8 membered    nonaromatic monocyclic ring having 0-1 heteroatoms independently    selected from O, N, and S.

Also included within this invention are pharmaceutically acceptablesalts of all the compounds of formula I, including but not limited to,the compounds of Table 1.

Compounds of this invention include those described generally above, andare further illustrated by the classes, subclasses, and speciesdisclosed herein. As used herein, the following definitions shall applyunless otherwise indicated. For purposes of this invention, the chemicalelements are identified in accordance with the Periodic Table of theElements, CAS version, Handbook of Chemistry and Physics, 75^(th) Ed.Additionally, general principles of organic chemistry are described in“Organic Chemistry”, Thomas Sorrell, University Science Books,Sausalito: 1999, and “March's Advanced Organic Chemistry”, 5^(th) Ed.,Ed.: Smith, M. B. and March, J., John Wiley & Sons, New York: 2001, theentire contents of which are hereby incorporated by reference.

As described herein, a specified number range of atoms includes anyinteger therein. For example, a group having from 1-4 atoms could have1, 2, 3, or 4 atoms.

As described herein, compounds of the invention may optionally besubstituted with one or more substituents, such as are illustratedgenerally above, or as exemplified by particular classes, subclasses,and species of the invention. It will be appreciated that the phrase“optionally substituted” is used interchangeably with the phrase“substituted or unsubstituted.” In general, the term “substituted”,whether preceded by the term “optionally” or not, refers to thereplacement of hydrogen radicals in a given structure with the radicalof a specified substituent. Unless otherwise indicated, an optionallysubstituted group may have a substituent at each substitutable positionof the group, and when more than one position in any given structure maybe substituted with more than one substituent selected from a specifiedgroup, the substituent may be either the same or different at everyposition. Combinations of substituents envisioned by this invention arepreferably those that result in the formation of stable or chemicallyfeasible compounds.

The term “stable”, as used herein, refers to compounds that are notsubstantially altered when subjected to conditions to allow for theirproduction, detection, recovery, purification, and use for one or moreof the purposes disclosed herein. In some embodiments, a stable compoundor chemically feasible compound is one that is not substantially alteredwhen kept at a temperature of 40° C. or less, in the absence of moistureor other chemically reactive conditions, for at least a week.

The term “aliphatic” or “aliphatic group”, as used herein, means astraight-chain (i.e., unbranched) or branched, substituted orunsubstituted hydrocarbon chain that is completely saturated or thatcontains one or more units of unsaturation that has a single point ofattachment to the rest of the molecule. Unless otherwise specified,aliphatic groups contain 1-20 aliphatic carbon atoms. In someembodiments, aliphatic groups contain 1-10 aliphatic carbon atoms. Inother embodiments, aliphatic groups contain 1-8 aliphatic carbon atoms.In still other embodiments, aliphatic groups contain 1-6 aliphaticcarbon atoms, and in yet other embodiments aliphatic groups contain 1-4aliphatic carbon atoms. Suitable aliphatic groups include, but are notlimited to, linear or branched, substituted or unsubstituted alkyl,alkenyl, or alkynyl groups. Specific examples include, but are notlimited to, methyl, ethyl, isopropyl, n-propyl, sec-butyl, vinyl,n-butenyl, ethynyl, and tert-butyl.

The term “cycloaliphatic” (or “carbocycle” or “carbocyclyl” or“cycloalkyl”) refers to a monocyclic C₃-C₈ hydrocarbon or bicyclicC₈-C₁₂ hydrocarbon that is completely saturated or that contains one ormore units of unsaturation, but which is not aromatic, that has a singlepoint of attachment to the rest of the molecule wherein any individualring in said bicyclic ring system has 3-7 members. Suitablecycloaliphatic groups include, but are not limited to, cycloalkyl andcycloalkenyl groups. Specific examples include, but are not limited to,cyclohexyl, cyclopropenyl, and cyclobutyl.

The term “heterocycle”, “heterocyclyl”, or “heterocyclic” as used hereinmeans non-aromatic, monocyclic, bicyclic, or tricyclic ring systems inwhich one or more ring members are an independently selected heteroatom.In some embodiments, the “heterocycle”, “heterocyclyl”, or“heterocyclic” group has three to fourteen ring members in which one ormore ring members is a heteroatom independently selected from oxygen,sulfur, nitrogen, or phosphorus, and each ring in the system contains 3to 7 ring members.

Suitable heterocycles include, but are not limited to,3-1H-benzimidazol-2-one, 3-(1-alkyl)-benzimidazol-2-one,2-tetrahydrofuranyl, 3-tetrahydrofuranyl, 2-tetrahydrothiophenyl,3-tetrahydrothiophenyl, 2-morpholino, 3-morpholino, 4-morpholino,2-thiomorpholino, 3-thiomorpholino, 4-thiomorpholino, 1-pyrrolidinyl,2-pyrrolidinyl, 3-pyrrolidinyl, 1-tetrahydropiperazinyl,2-tetrahydropiperazinyl, 3-tetrahydropiperazinyl, 1-piperidinyl,2-piperidinyl, 3-piperidinyl, 1-pyrazolinyl, 3-pyrazolinyl,4-pyrazolinyl, 5-pyrazolinyl, 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-piperidinyl, 2-thiazolidinyl, 3-thiazolidinyl,4-thiazolidinyl, 1-imidazolidinyl, 2-imidazolidinyl, 4-imidazolidinyl,5-imidazolidinyl, indolinyl, tetrahydroquinolinyl,tetrahydroisoquinolinyl, benzothiolane, benzodithiane, and1,3-dihydro-imidazol-2-one.

Cyclic groups, (e.g., cycloaliphatic and heterocycles), can be linearlyfused, bridged, or spirocyclic.

The term “heteroatom” means one or more of oxygen, sulfur, nitrogen, orphosphorus, (including, any oxidized form of nitrogen, sulfur, orphosphorus; the quaternized form of any basic nitrogen or; asubstitutable nitrogen of a heterocyclic ring, for example N (as in3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl) or NR⁺ (as inN-substituted pyrrolidinyl).

The term “unsaturated”, as used herein, means that a moiety has one ormore units of unsaturation.

The term “nonaromatic”, as used herein, describes rings that are eithersaturated or partially unsaturated.

The term “aromatic”, as used herein, describes rings that are fullyunsaturated.

The term “alkoxy”, or “thioalkyl”, as used herein, refers to an alkylgroup, as previously defined, attached to the principal carbon chainthrough an oxygen (“alkoxy”) or sulfur (“thioalkyl”) atom.

The terms “haloalkyl”, “haloalkenyl”, “haloaliphatic”, and “haloalkoxy”mean alkyl, alkenyl or alkoxy, as the case may be, substituted with oneor more halogen atoms. The terms “halogen”, “halo”, and “hal” mean F,Cl, Br, or I.

The term “aryl” used alone or as part of a larger moiety as in“aralkyl”, “aralkoxy”, or “aryloxyalkyl”, refers to monocyclic,bicyclic, and tricyclic ring systems having a total of five to fourteenring members, wherein at least one ring in the system is aromatic andwherein each ring in the system contains 3 to 7 ring members. The term“aryl” may be used interchangeably with the term “aryl ring”. The term“aryl” also refers to heteroaryl ring systems as defined hereinbelow.

The term “heteroaryl”, used alone or as part of a larger moiety as in“heteroaralkyl” or “heteroarylalkoxy”, refers to monocyclic, bicyclic,and tricyclic ring systems having a total of five to fourteen ringmembers, wherein at least one ring in the system is aromatic, at leastone ring in the system contains one or more heteroatoms, and whereineach ring in the system contains 3 to 7 ring members. The term“heteroaryl” may be used interchangeably with the term “heteroaryl ring”or the term “heteroaromatic”. Suitable heteroaryl rings include, but arenot limited to, 2-furanyl, 3-furanyl, N-imidazolyl, 2-imidazolyl,4-imidazolyl, 5-imidazolyl, benzimidazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-oxazolyl, 4-oxazolyl, 5-oxazolyl, N-pyrrolyl,2-pyrrolyl, 3-pyrrolyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidinyl,4-pyrimidinyl, 5-pyrimidinyl, pyridazinyl (e.g., 3-pyridazinyl),2-thiazolyl, 4-thiazolyl, 5-thiazolyl, tetrazolyl (e.g., 5-tetrazolyl),triazolyl (e.g., 2-triazolyl and 5-triazolyl), 2-thienyl, 3-thienyl,benzofuryl, benzothiophenyl, indolyl (e.g., 2-indolyl), pyrazolyl (e.g.,2-pyrazolyl), isothiazolyl, 1,2,3-oxadiazolyl, 1,2,5-oxadiazolyl,1,2,4-oxadiazolyl, 1,2,3-triazolyl, 1,2,3-thiadiazolyl,1,3,4-thiadiazolyl, 1,2,5-thiadiazolyl, purinyl, pyrazinyl,1,3,5-triazinyl, quinolinyl (e.g., 2-quinolinyl, 3-quinolinyl,4-quinolinyl), and isoquinolinyl (e.g., 1-isoquinolinyl,3-isoquinolinyl, or 4-isoquinolinyl).

The term “protecting group” and “protective group” as used herein, areinterchangeable and refer to an agent used to temporarily block one ormore desired reactive sites in a multifunctional compound. In certainembodiments, a protecting group has one or more, or preferably all, ofthe following characteristics: a) is added selectively to a functionalgroup in good yield to give a protected substrate that is b) stable toreactions occurring at one or more of the other reactive sites; and c)is selectively removable in good yield by reagents that do not attackthe regenerated, deprotected functional group. Exemplary protectinggroups are detailed in Greene, T. W., Wuts, P. G. in “Protective Groupsin Organic Synthesis”, Third Edition, John Wiley & Sons, New York: 1999(and other editions of the book), the entire contents of which arehereby incorporated by reference. The term “nitrogen protecting group”,as used herein, refers to an agents used to temporarily block one ormore desired nitrogen reactive sites in a multifunctional compound.Preferred nitrogen protecting groups also possess the characteristicsexemplified above, and certain exemplary nitrogen protecting groups arealso detailed in Chapter 7 in Greene, T. W., Wuts, P. G. in “ProtectiveGroups in Organic Synthesis”, Third Edition, John Wiley & Sons, NewYork: 1999, the entire contents of which are hereby incorporated byreference.

In some embodiments, an alkyl or aliphatic chain can be optionallyinterrupted with another atom or group. This means that a methylene unitof the alkyl or aliphatic chain is optionally replaced with said otheratom or group. Examples of such atoms or groups would include, but arenot limited to, —NR—, —O—, —S—, —CO₂—, —OC(O)—, —C(O)CO—, —C(O)—,—C(O)NR—, —C(═N—CN), —NRCO—, —NRC(O)O—, —SO₂NR—, —NRSO₂—, —NRC(O)NR—,—OC(O)NR—, —NRSO₂NR—, —SO—, or —SO₂—, wherein R is defined herein.Unless otherwise specified, the optional replacements form a chemicallystable compound. Optional interruptions can occur both within the chainand at either end of the chain; i.e., both at the point of attachmentand/or also at the terminal end. Two optional replacements can also beadjacent to each other within a chain so long as it results in achemically stable compound. The optional interruptions or replacementscan also completely replace all of the carbon atoms in a chain. Forexample, a C₃ aliphatic can be optionally interrupted or replaced by—NR—, —C(O)—, and —NR— to form —NRC(O)NR— (a urea).

Unless otherwise specified, if the replacement or interruption occurs atthe terminal end, the replacement atom is bound to an H on the terminalend. For example, if —CH₂CH₂CH₃ were optionally interrupted with —O—,the resulting compound could be —OCH₂CH₃, —CH₂OCH₃, or —CH₂CH₂OH.

Unless otherwise stated, structures depicted herein are also meant toinclude all isomeric (e.g., enantiomeric, diastereomeric, and geometric(or conformational)) forms of the structure; for example, the R and Sconfigurations for each asymmetric center, (Z) and (E) double bondisomers, and (Z) and (E) conformational isomers. Therefore, singlestereochemical isomers as well as enantiomeric, diastereomeric, andgeometric (or conformational) mixtures of the present compounds arewithin the scope of the invention.

Unless otherwise stated, all tautomeric forms of the compounds of theinvention are within the scope of the invention.

Unless otherwise stated, a substituent can freely rotate around anyrotatable bonds. For example, a substituent drawn as

also represents

Additionally, unless otherwise stated, structures depicted herein arealso meant to include compounds that differ only in the presence of oneor more isotopically enriched atoms. For example, compounds having thepresent structures except for the replacement of hydrogen by deuteriumor tritium, or the replacement of a carbon by a ¹³C- or ¹⁴C-enrichedcarbon are within the scope of this invention. Such compounds areuseful, for example, as analytical tools or probes in biological assays.

The following abbreviations are used:

-   PG protecting group-   LG leaving group-   DCM dichloromethane-   Ac acetyl-   DMF dimethylformamide-   EtOAc ethyl acetate-   DMSO dimethyl sulfoxide-   MeCN acetonitrile-   TCA trichloroacetic acid-   ATP adenosine triphosphate-   EtOH ethanol-   Ph phenyl-   Me methyl-   Et ethyl-   Bu butyl-   DEAD diethylazbdicarboxylate-   HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-   BSA bovine serum albumin-   DTT dithiothreitol-   MOPS 4-morpholinepropanesulfonic acid-   NMR nuclear magnetic resonance-   HPLC high performance liquid chromatography-   LCMS liquid chromatography-mass spectrometry-   TLC thin layer chromatography-   Rt retention time

In some embodiments of this invention, G is —C(R)₂—. In otherembodiments, G is O.

In some embodiments, R¹ is H.

In some embodiments, Ring A is selected from

In some embodiments, Ring B is a 5-6 membered aromatic monocyclic ringcontaining 0-3 heteroatoms selected from O, N, and S. In someembodiments, Ring B is a 5-membered ring. In other embodiments, Ring Bis a 6-membered ring. In some embodiments, Ring B is fused to Ring B′.In some embodiments, Ring B′ is a 5-6 membered aromatic monocyclic ringcontaining 0-3 heteroatoms selected from O, N, and S. In all of theseembodiments, Ring B is optionally substituted with 0-5 J^(B) and Ring B′is optionally substituted with 0-4 J^(B′).

In some embodiments, J^(A) is H, C₁₋₆ aliphatic, C₃₋₆cycloaliphatic,halo(C₁₋₄ aliphatic), —O(haloC₁₋₄ aliphatic), C₃₋₆heterocyclyl, halo,NO₂, CN, OH, OR, SH, SR, NH₂, NHR, N(R)₂, COH, COR, CONH₂, CONHR, CONR₂,NHCOR, NRCOR, NHCONH₂, NHCONHR, NHCON(R)₂, SO₂NH₂, SO₂NHR, SO₂N(R)₂,NHSO₂R, or NRSO₂R. In some embodiments, J^(A) is H.

In some embodiments, J^(B) is H, C₁₋₆ aliphatic, C₃₋₆cycloaliphatic,halo(C₁₋₄ aliphatic), —O(haloC₁₋₄ aliphatic), C₃₋₆heterocyclyl, halo,NO₂, CN, OH, OR, SH, SR, NH₂, NHR, N(R)₂, COH, COR, CONH₂, CONHR, CONR₂,NHCOR, NRCOR, NHCONH₂, NHCONHR, NHCON(R)₂, SO₂NH₂, SO₂NHR, SO₂N(R)₂,NHSO₂R, or NRSO₂R.

In other embodiments, J^(B′) is H, C₁₋₆ aliphatic, C₃₋₆cycloaliphatic,halo(Cl₄ aliphatic), —O(haloC₁₋₄ aliphatic), C₃₋₆ heterocyclyl, halo,NO₂, CN, OH, OR, SH, SR, NH₂, NHR, N(R)₂, COH, COR, CONH₂, CONHR, CONR₂,NHCOR, NRCOR, NHCONH₂, NHCONHR, NHCON(R)₂, SO₂NH₂, SO₂NHR, SO₂N(R)₂,NHSO₂R, or NRSO₂R.

In some embodiments, L is C₀₋₃alkyl optionally substituted with 0-3J^(L). In other embodiments, L is C₁₋₃alkyl optionally substituted with0-3 J^(L). In other embodiments, L is —CH(CH₃)—.

In some embodiments, Ring B is phenyl, optionally substituted with 0-5J^(B).

In some embodiments, the variables are as described in the compoundsrepresented in Table 1.

In some embodiments, the compounds of this invention are as representedin Table 1:

TABLE 1

General Synthetic Methodology

The compounds of this invention may be prepared in general by methodssuch as those depicted in the general schemes below, and the preparativeexamples that follow. Unless otherwise indicated, all variables in thefollowing schemes are as defined herein.

Scheme 1 above shows a general synthetic route for preparing compoundsof this invention where ring A is connected to the thiophene nucleus viaa nitrogen atom. Michael acceptor 1 (prepared using method similar tothe one reported in Synthesis, (10), 847-850, 1984) is reacted in aconjugate addition with

wherein ring A is a nitrogen-containing ring, to generate 2. The3-hydroxy group of 2 is then derivatised with appropriate functionalgroups under Mitsunobu conditions to give 3. Finally the ester moiety in3 is transformed into an amide under suitable amide formation conditionsto give the compounds of this invention.

Scheme 2 above shows a general synthetic route for preparing certainring A derivatives. Cyanamide 5 is treated with an aldehyde and ahydrazine (in a similar manner to the one reported in Synthesis, (3),222-223, 1983) to give aminotriazole 6. This can then undergo furtherfunctionalisation as outlined in Scheme 1 to give compounds of thisinvention.

Accordingly, this invention also provides a process for preparing acompound of this invention.

One embodiment of this invention provides a process for preparing acompound of formula I:

wherein G is O, R¹ is H, and Ring A, Ring B, J^(A), J^(B), and L are asdefined herein,comprising reacting a compound of formula 3

wherein G is O, R¹ is C₁₋₆aliphatic, and Ring A, Ring B, J^(A), J^(B),and L are as defined herein,under suitable amide forming conditions to form the compound of formulaI. Suitable amide forming conditions are known to one of skill in theart and can be found in “March's Advanced Organic Chemistry”. An exampleof a suitable amide forming condition includes, but is not limited to,heating the methyl carboxylate in the presence of NH₃/MeOH.

One embodiment of this invention provides a process for preparing acompound of formula 3:

wherein G is O and Ring A, Ring B, J^(A), J^(B) and L are as definedherein,comprising reacting a compound of formula 2:

with

wherein LG is a suitable leaving group and L, Ring B, and J^(B) are asdefined herein; under suitable O—C bond coupling conditions to form thecompound of formula 3.

Another embodiment provides a process for preparing a compound offormula 2:

comprising adding

wherein ring A is an aromatic ring containing a nitrogen atom capable ofnucleophilic attack and JA is as defined herein; to a compound offormula 1:

via suitable conjugate addition conditions, to form the compound offormula 2. An example of an aromatic ring containing a nitrogen atomcapable of nucleophilic attack is

Suitable conjugate addition conditions include, but are not limited to,combining 2 equivalents of

with the compound of formula 8 in DCM at room temperature; combining1.15 equivalents of

with the compound of formula 8 in acetonitrile/DMF at 110 degreesCelsius overnight.

Another embodiment provides a process for preparing a compound offormula 6:

comprising treating a compound of formula 5:

with an aldehyde and a hydrazine (in a similar manner to the onereported in Synthesis, (3), 222-223, 1983) to form the compound offormula 6.

In some embodiments,

is the compound of formula 6.

The present invention provides compounds that are useful for thetreatment of diseases, disorders, and conditions including, but notlimited to, autoimmune diseases, inflammatory diseases, proliferativeand hyperproliferative diseases, immunologically-mediated diseases, bonediseases, metabolic diseases, neurological and neurodegenerativediseases, cardiovascular diseases, hormone related diseases, allergies,asthma, and Alzheimer's disease. Another aspect of this inventionprovides compounds that are inhibitors of protein kinases, and thus areuseful for the treatment of the diseases, disorders, and conditions,along with other uses described herein. In another aspect of the presentinvention, pharmaceutically acceptable compositions are provided,wherein these compositions comprise any of the compounds as describedherein, and optionally comprise a pharmaceutically acceptable carrier,adjuvant or vehicle. In certain embodiments, these compositionsoptionally further comprise one or more additional therapeutic agents.

It will also be appreciated that certain of the compounds of presentinvention can exist in free form for treatment, or where appropriate, asa pharmaceutically acceptable salt or pharmaceutically acceptablederivative thereof.

As used herein, a “pharmaceutically acceptable derivative” is an adductor derivative which, upon administration to a patient in need, iscapable of providing, directly or indirectly, a compound as otherwisedescribed herein, or a metabolite or residue thereof. Examples ofpharmaceutically acceptable derivatives include, but are not limited to,esters and salts of such esters.

As used herein, the term “pharmaceutically acceptable salt” refers tosalts of a compound which are, within the scope of sound medicaljudgment, suitable for use in contact with the tissues of humans andlower animals without undue toxicity, irritation, allergic response andthe like, and are commensurate with a reasonable benefit/risk ratio.

Pharmaceutically acceptable salts are well known in the art. Forexample, S. M. Berge et al., describe pharmaceutically acceptable saltsin detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, incorporatedherein by reference. Pharmaceutically acceptable salts of the compoundsof this invention include those derived from suitable inorganic andorganic acids and bases. These salts can be prepared in situ during thefinal isolation and purification of the compounds. Acid addition saltscan be prepared by 1) reacting the purified compound in its free-basedform with a suitable organic or inorganic acid and 2) isolating the saltthus formed.

Examples of pharmaceutically acceptable, nontoxic acid addition saltsare salts of an amino group formed with inorganic acids such ashydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid andperchloric acid or with organic acids such as acetic acid, oxalic acid,maleic acid, tartaric acid, citric acid, succinic acid or malonic acidor by using other methods used in the art such as ion exchange. Otherpharmaceutically acceptable salts include adipate, alginate, ascorbate,aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate,camphorate, camphorsulfonate, citrate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate,glucoheptonate, glycerophosphate, glycolate, gluconate, hemisulfate,heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate,lactobionate, lactate, laurate, lauryl sulfate, malate, maleate,malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate,oleate, oxalate, palmitate, palmoate, pectinate, persulfate,3-phenylpropionate, phosphate, picrate, pivalate, propionate,salicylate, stearate, succinate, sulfate, tartrate, thiocyanate,p-toluenesulfonate, undecanoate, valerate salts, and the like. Saltsderived from appropriate bases include alkali metal, alkaline earthmetal, ammonium and N⁺(C₁₋₄alkyl)₄ salts. This invention also envisionsthe quaternization of any basic nitrogen-containing groups of thecompounds disclosed herein. Water or oil-soluble or dispersible productsmay be obtained by such quaternization.

Base addition salts can be prepared by 1) reacting the purified compoundin its acid form with a suitable organic or inorganic base and 2)isolating the salt thus formed. Base addition salts include alkali oralkaline earth metal salts. Representative alkali or alkaline earthmetal salts include sodium, lithium, potassium, calcium, magnesium, andthe like. Further pharmaceutically acceptable salts include, whenappropriate, nontoxic ammonium, quaternary ammonium, and amine cationsformed using counterions such as halide, hydroxide, carboxylate,sulfate, phosphate, nitrate, loweralkyl sulfonate and aryl sulfonate.Other acids and bases, while not in themselves pharmaceuticallyacceptable, may be employed in the preparation of salts useful asintermediates in obtaining the compounds of the invention and theirpharmaceutically acceptable acid or base addition salts.

As described herein, the pharmaceutically acceptable compositions of thepresent invention additionally comprise a pharmaceutically acceptablecarrier, adjuvant, or vehicle, which, as used herein, includes any andall solvents, diluents, or other liquid vehicle, dispersion orsuspension aids, surface active agents, isotonic agents, thickening oremulsifying agents, preservatives, solid binders, lubricants and thelike, as suited to the particular dosage form desired. Remington'sPharmaceutical Sciences, Sixteenth Edition, E. W. Martin (MackPublishing Co., Easton, Pa., 1980) discloses various carriers used informulating pharmaceutically acceptable compositions and knowntechniques for the preparation thereof. Except insofar as anyconventional carrier medium is incompatible with the compounds of theinvention, such as by producing any undesirable biological effect orotherwise interacting in a deleterious manner with any othercomponent(s) of the pharmaceutically acceptable composition, its use iscontemplated to be within the scope of this invention.

Some examples of materials which can serve as pharmaceuticallyacceptable carriers-include, but are not limited to, ion exchangers,alumina, aluminum stearate, lecithin, serum proteins, such as humanserum albumin, buffer substances such as phosphates, glycine, sorbicacid, or potassium sorbate, partial glyceride mixtures of saturatedvegetable fatty acids, water, salts or electrolytes, such as protaminesulfate, disodium hydrogen phosphate, potassium hydrogen phosphate,sodium chloride, zinc salts, colloidal silica, magnesium trisilicate,polyvinyl pyrrolidone, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, wool fat, sugars such aslactose, glucose and sucrose; starches such as corn starch and potatostarch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethyl cellulose and cellulose acetate; powdered tragacanth;malt; gelatin; talc; excipients such as cocoa butter and suppositorywaxes; oils such as peanut oil, cottonseed oil; safflower oil; sesameoil; olive oil; corn oil and soybean oil; glycols; such a propyleneglycol or polyethylene glycol; esters such as ethyl oleate and ethyllaurate; agar; buffering agents such as magnesium hydroxide and aluminumhydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer'ssolution; ethyl alcohol, and phosphate buffer solutions, as well asother non-toxic compatible lubricants such as sodium lauryl sulfate andmagnesium stearate, as well as coloring agents, releasing agents,coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the composition,according to the judgment of the formulator.

One aspect of this invention provides a method for the treatment orlessening the severity of a disease selected from an autoimmune disease,an inflammatory disease, a proliferative or hyperproliferative disease,such as cancer, an immunologically-mediated disease, a bone disease, ametabolic disease, a neurological or neurodegenerative disease, acardiovascular disease, allergies, asthma, Alzheimer's disease, or ahormone related disease, comprising administering an effective amount ofa compound, or a pharmaceutically acceptable composition comprising acompound, to a subject in need thereof. The term “cancer” includes, butis not limited to the following cancers: breast; ovary; cervix;prostate; testis, genitourinary tract; esophagus; larynx, glioblastoma;neuroblastoma; stomach; skin, keratoacanthoma; lung, epidermoidcarcinoma, large cell carcinoma, small cell carcinoma, lungadenocarcinoma; bone; colon, adenoma; pancreas, adenocarcinoma; thyroid,follicular carcinoma, undifferentiated carcinoma, papillary carcinoma;seminoma; melanoma; sarcoma; bladder carcinoma; liver carcinoma andbiliary passages; kidney carcinoma; myeloid disorders; lymphoiddisorders, Hodgkin's, hairy cells; buccal cavity and pharynx (oral),lip, tongue, mouth, pharynx; small intestine; colon-rectum, largeintestine, rectum; brain and central nervous system; and leukemia.

In certain embodiments, an “effective amount” of the compound orpharmaceutically acceptable composition is that amount effective inorder to treat said disease. The compounds and compositions, accordingto the method of the present invention, may be administered using anyamount and any route of administration effective for treating orlessening the severity of said disease. In some embodiments, saiddisease is selected from a proliferative disorder, a neurodegenerativedisorder, an autoimmune disorder, and inflammatory disorder, and animmunologically-mediated disorder. In some embodiments, said disease isa proliferative disorder. In some embodiments, cancer.

In other embodiments of this invention, said disease is a protein-kinasemediated condition. In some embodiments, said protein kinase in PLK.

The term “protein kinase-mediated condition”, as used herein means anydisease or other deleterious condition in which a protein kinase plays arole. Such conditions include, without limitation, autoimmune diseases,inflammatory diseases, proliferative and hyperproliferative diseases,immunologically-mediated diseases, bone diseases, metabolic diseases,neurological and neurodegenerative diseases, cardiovascular diseases,hormone related diseases, allergies, asthma, and Alzheimer's disease.

The term “PLK-mediated condition”, as used herein means any disease orother deleterious condition in which PLK plays a role. Such conditionsinclude, without limitation, a proliferative disorder, such as cancer, aneurodegenerative disorder, an autoimmune disorder, and inflammatorydisorder, and an immunologically-mediated disorder.

In some embodiments; the compounds and compositions of the invention areinhibitors of protein kinases. As inhibitors of protein kinases, thecompounds and compositions of this invention are particularly useful fortreating or lessening the severity of a disease, condition, or disorderwhere a protein kinase is implicated in the disease, condition, ordisorder. In one aspect, the present invention provides a method fortreating or lessening the severity of a disease, condition, or disorderwhere a protein kinase is implicated in the disease state. In anotheraspect, the present invention provides a method for treating orlessening the severity of a disease, condition, or disorder whereinhibition of enzymatic activity is implicated in the treatment of thedisease. In another aspect, this invention provides a method fortreating or lessening the severity of a disease, condition, or disorderwith compounds that inhibit enzymatic activity by binding to the proteinkinase. In some embodiments, said protein kinase is PLK.

The activity of the compounds as protein kinase inhibitors may beassayed in vitro, in vivo or in a cell line. In vitro assays includeassays that determine inhibition of either the kinase activity or ATPaseactivity of the activated kinase. Alternate in vitro assays quantitatethe ability of the inhibitor to bind to the protein kinase and may bemeasured either by radiolabelling the inhibitor prior to binding,isolating the inhibitor/kinase complex and determining the amount ofradiolabel bound, or by running a competition experiment where newinhibitors are incubated with the kinase bound to known radioligands.

The protein kinase inhibitors or pharmaceutical salts thereof may beformulated into pharmaceutical compositions for administration toanimals or humans. These pharmaceutical compositions, which comprise anamount of the protein inhibitor effective to treat or prevent a proteinkinase-mediated condition and a pharmaceutically acceptable carrier, areanother embodiment of the present invention. In some embodiments, saidprotein kinase-mediated condition is a PLK-mediated condition. In someembodiments, a PLK1-mediated condition.

The exact amount of compound required for treatment will vary fromsubject to subject, depending on the species, age, and general conditionof the subject, the severity of the infection, the particular agent, itsmode of administration, and the like. The compounds of the invention arepreferably formulated in dosage unit form for ease of administration anduniformity of dosage. The expression “dosage unit form” as used hereinrefers to a physically discrete unit of agent appropriate for thepatient to be treated. It will be understood, however, that the totaldaily usage of the compounds and compositions of the present inventionwill be decided by the attending physician within the scope of soundmedical judgment. The specific effective dose level for any particularpatient or organism will depend upon a variety of factors including thedisorder being treated and the severity of the disorder; the activity ofthe specific compound employed; the specific composition employed; theage, body weight, general health, sex and diet of the patient; the timeof administration, route of administration, and rate of excretion of thespecific compound employed; the duration of the treatment; drugs used incombination or coincidental with the specific compound employed, andlike factors well known in the medical arts. The term “patient”, as usedherein, means an animal, preferably a mammal, and most preferably ahuman.

The pharmaceutically acceptable compositions of this invention can beadministered to humans and other animals orally, rectally, parenterally,intracisternally, intravaginally, intraperitoneally, topically (as bypowders, ointments, or drops), bucally, as an oral or nasal spray, orthe like, depending on the severity of the infection being treated. Incertain embodiments, the compounds of the invention may be administeredorally or parenterally at dosage levels of about 0.01 mg/kg to about 50mg/kg and preferably from about 1 mg/kg to about 25 mg/kg, of subjectbody weight per day, one or more times a day, to obtain the desiredtherapeutic effect.

Liquid dosage forms for oral administration include, but are not limitedto, pharmaceutically acceptable emulsions, microemulsions, solutions,suspensions, syrups and elixirs. In addition to the active compounds,the liquid dosage forms may contain inert diluents commonly used in theart such as, for example, water or other solvents, solubilizing agentsand emulsifiers such as ethyl alcohol, isopropyl alcohol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butylene glycol, dimethylformamide, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor, and sesame oils),glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fattyacid esters of sorbitan, and mixtures thereof. Besides inert diluents,the oral compositions can also include adjuvants such as wetting agents,emulsifying and suspending agents, sweetening, flavoring, and perfumingagents.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

The injectable formulations can be sterilized, for example, byfiltration through a bacterial-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

In order to prolong the effect of a compound of the present invention,it is often desirable to slow the absorption of the compound fromsubcutaneous or intramuscular injection. This may be accomplished by theuse of a liquid suspension of crystalline or amorphous material withpoor water solubility. The rate of absorption of the compound thendepends upon its rate of dissolution that, in turn, may depend uponcrystal size and crystalline form. Alternatively, delayed absorption ofa parenterally administered compound form is accomplished by dissolvingor suspending the compound in an oil vehicle. Injectable depot forms aremade by forming microencapsule matrices of the compound in biodegradablepolymers such as polylactide-polyglycolide. Depending upon the ratio ofcompound to polymer and the nature of the particular polymer employed,the rate of compound release can be controlled. Examples of otherbiodegradable polymers include poly(orthoesters) and poly(anhydrides).Depot injectable formulations are also prepared by entrapping thecompound in liposomes or microemulsions that are compatible with bodytissues.

Compositions for rectal or vaginal administration are preferablysuppositories which can be prepared by mixing the compounds of thisinvention with suitable non-irritating excipients or carriers such ascocoa butter, polyethylene glycol or a suppository wax which are solidat ambient temperature but liquid at body temperature and therefore meltin the rectum or vaginal cavity and release the active compound.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activecompound is mixed with at least one inert, pharmaceutically acceptableexcipient or carrier such as sodium citrate or dicalcium phosphateand/or a) fillers or extenders such as starches, lactose, sucrose,glucose, mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents such as paraffin, f) absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, h) absorbents such as kaolinand bentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets and pills, thedosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like. The solid dosage forms of tablets, dragees, capsules, pills,and granules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally contain opacifying agents and can also be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions that can be usedinclude polymeric substances and waxes. Solid compositions of a similartype may also be employed as fillers in soft and hard-filled gelatincapsules using such excipients as lactose or milk sugar as well as highmolecular weight polethylene glycols and the like.

The active compounds can also be in microencapsulated form with one ormore excipients as noted above. The solid dosage forms of tablets,dragees, capsules, pills, and granules can be prepared with coatings andshells such as enteric coatings, release controlling coatings and othercoatings well known in the pharmaceutical formulating art. In such soliddosage forms the active compound may be admixed with at least one inertdiluent such as sucrose, lactose or starch. Such dosage forms may alsocomprise, as is normal practice, additional substances other than inertdiluents, e.g., tableting lubricants and other tableting aids such amagnesium stearate and microcrystalline cellulose. In the case ofcapsules, tablets and pills, the dosage forms may also comprisebuffering agents. They may optionally contain opacifying agents and canalso be of a composition that they release the active ingredient(s)only, or preferentially, in a certain part of the intestinal tract,optionally, in a delayed manner. Examples of embedding compositions thatcan be used include polymeric substances and waxes.

Dosage forms for topical or transdermal administration of a compound ofthis invention include ointments, pastes, creams, lotions, gels,powders, solutions, sprays, inhalants or patches. The active componentis admixed under sterile conditions with a pharmaceutically acceptablecarrier and any needed preservatives or buffers as may be required.Ophthalmic formulation, eardrops, and eye drops are also contemplated asbeing within the scope of this invention. Additionally, the presentinvention contemplates the use of transdermal patches, which have theadded advantage of providing controlled delivery of a compound to thebody. Such dosage forms can be made by dissolving or dispensing thecompound in the proper medium. Absorption enhancers can also be used toincrease the flux of the compound across the skin. The rate can becontrolled by either providing a rate controlling membrane or bydispersing the compound in a polymer matrix or gel.

In addition to the compounds of this invention, pharmaceuticallyacceptable derivatives or prodrugs of the compounds of this inventionmay also be employed in compositions to treat or prevent theabove-identified disorders.

A “pharmaceutically acceptable derivative or prodrug” means anypharmaceutically acceptable ester, salt of an ester or other derivativeof a compound of this invention which, upon administration to arecipient, is capable of providing, either directly or indirectly, acompound of this invention or an inhibitorily active metabolite orresidue thereof. Particularly favoured derivatives or prodrugs are thosethat increase the bioavailability of the compounds of this inventionwhen such compounds are administered to a patient (e.g., by allowing anorally administered compound to be more readily absorbed into the blood)or which enhance delivery of the parent compound to a biologicalcompartment (e.g., the brain or lymphatic system) relative to the parentspecies.

Pharmaceutically acceptable prodrugs of the compounds of this inventioninclude, without limitation, esters, amino acid esters, phosphateesters, metal salts and sulfonate esters.

Pharmaceutically acceptable carriers that may be used in thesepharmaceutical compositions include, but are not limited to, ionexchangers, alumina, aluminum stearate, lecithin, serum proteins, suchas human serum albumin, buffer substances such as phosphates, glycine,sorbic acid, potassium sorbate, partial glyceride mixtures of saturatedvegetable fatty acids, water, salts or electrolytes, such as protaminesulfate, disodium hydrogen phosphate, potassium hydrogen phosphate,sodium chloride, zinc salts, colloidal silica, magnesium trisilicate,polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol,sodium carboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat.

The compositions of the present invention may be administered orally,parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir. The term “parenteral”as used herein includes, but is not limited to, subcutaneous,intravenous, intramuscular, intra-articular, intra-synovial,intrasternal, intrathecal, intrahepatic, intralesional and intracranialinjection or infusion techniques. Preferably, the compositions areadministered orally, intraperitoneally or intravenously.

Sterile injectable forms of the compositions of this invention may beaqueous or oleaginous suspension. These suspensions may be formulatedaccording to techniques known in the art using suitable dispersing orwetting agents and suspending agents. The sterile injectable preparationmay also be a sterile injectable solution or suspension in a non-toxicparenterally-acceptable diluent or solvent, for example as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose, any bland fixed oilmay be employed including synthetic mono- or di-glycerides. Fatty acids,such as oleic acid and its glyceride derivatives are useful in thepreparation of injectables, as are natural pharmaceutically-acceptableoils, such as olive-oil or castor oil, especially in theirpolyoxyethylated versions. These oil solutions or suspensions may alsocontain a long-chain alcohol diluent or dispersant, such ascarboxymethyl cellulose or similar dispersing agents which are commonlyused in the formulation of pharmaceutically acceptable dosage formsincluding emulsions and suspensions. Other commonly used surfactants,such as Tweens, Spans and other emulsifying agents or bioavailabilityenhancers which are commonly used in the manufacture of pharmaceuticallyacceptable solid, liquid, or other dosage forms may also be used for thepurposes of formulation.

The pharmaceutical compositions of this invention may be orallyadministered in any orally acceptable dosage form including, but notlimited to, capsules, tablets, aqueous suspensions or solutions. In thecase of tablets for oral use, carriers commonly used include, but arenot limited to, lactose and corn starch. Lubricating agents, such asmagnesium stearate, are also typically added. For oral administration ina capsule form, useful diluents include lactose and dried cornstarch.When aqueous suspensions are required for oral use, the activeingredient is combined with emulsifying and suspending agents. Ifdesired, certain sweetening, flavoring or coloring agents may also beadded.

Alternatively, the pharmaceutical compositions of this invention may beadministered in the form of suppositories for rectal administration.These can be prepared by mixing the agent with a suitable non-irritatingexcipient which is solid at room temperature but liquid at rectaltemperature and therefore will melt in the rectum to release the drug.Such materials include, but are not limited to, cocoa butter, beeswaxand polyethylene glycols.

The pharmaceutical compositions of this invention may also beadministered topically, especially when the target of treatment includesareas or organs readily accessible by topical application, includingdiseases of the eye, the skin, or the lower intestinal tract. Suitabletopical formulations are readily prepared for each of these areas ororgans.

Topical application for the lower intestinal tract can be effected in arectal suppository formulation (see above) or in a suitable enemaformulation. Topically-transdermal patches may also be used.

For topical applications, the pharmaceutical compositions may beformulated in a suitable ointment containing the active componentsuspended or dissolved in one or more carriers. Carriers for topicaladministration of the compounds of this invention include, but are notlimited to, mineral oil, liquid petrolatum, white petrolatum, propyleneglycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax andwater. Alternatively, the pharmaceutical compositions can be formulatedin a suitable lotion or cream containing the active components suspendedor dissolved in one or more pharmaceutically acceptable carriers.Suitable carriers include, but are not limited to, mineral oil, sorbitanmonostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol,2-octyldodecanol, benzyl alcohol and water.

For ophthalmic use, the pharmaceutical compositions may be formulated asmicronized suspensions in isotonic, pH adjusted sterile saline, or,preferably, as solutions in isotonic, pH adjusted sterile saline, eitherwith or without a preservative such as benzylalkonium chloride.Alternatively, for ophthalmic uses, the pharmaceutical compositions maybe formulated in an ointment such as petrolatum.

The pharmaceutical compositions of this invention may also beadministered by nasal aerosol or inhalation. Such compositions areprepared according to techniques well-known in the art of pharmaceuticalformulation and may be prepared as solutions in saline, employing benzylalcohol or other suitable preservatives, absorption promoters to enhancebioavailability, fluorocarbons, and/or other conventional solubilizingor dispersing agents.

The amount of protein kinase inhibitor that may be combined with thecarrier materials to produce a single dosage form will vary dependingupon the host treated, the particular mode of administration.Preferably, the compositions should be formulated so that a dosage ofbetween 0.01-100 mg/kg body weight/day of the inhibitor can beadministered to a patient receiving these compositions.

It should also be understood that a specific dosage and treatmentregimen for any particular patient will depend upon a variety offactors, including the activity of the specific compound employed, theage, body weight, general health, sex, diet, time of administration,rate of excretion, drug combination, and the judgment of the treatingphysician and the severity of the particular disease being treated. Theamount of inhibitor will also depend upon the particular compound in thecomposition.

According to another embodiment, the invention provides methods fortreating or preventing a protein kinase-mediated condition (in someembodiments, a PLK-mediated condition) comprising the step ofadministering to a patient one of the above-described pharmaceuticalcompositions. The term “patient”, as used herein, means an animal,preferably a human.

Preferably, that method is used to treat or prevent a condition selectedfrom cancers such as cancers of the breast, colon, prostate, skin,pancreas, brain, genitourinary tract, lymphatic system, stomach, larynxand lung, including lung adenocarcinoma and small cell lung cancer;stroke, diabetes, myeloma, hepatomegaly, cardiomegaly, Alzheimer'sdisease, cystic fibrosis, and viral disease, or any specific diseasedescribed above.

Another aspect of the invention relates to inhibiting protein kinaseactivity in a patient, which method comprises administering to thepatient a compound of formula I or a composition comprising saidcompound.

Depending upon the particular protein kinase-mediated conditions to betreated or prevented, additional drugs, which are normally administeredto treat or prevent that condition, may be administered together withthe inhibitors of this invention. For example, chemotherapeutic agentsor other anti-proliferative agents may be combined with the proteinkinase inhibitors of this invention to treat proliferative diseases.

Those additional agents may be administered separately, as part of amultiple dosage regimen, from the protein kinase inhibitor-containingcompound or composition. Alternatively, those agents may be part of asingle dosage form, mixed together with the protein kinase inhibitor ina single composition.

In some embodiments, said protein kinase inhibitor is a PLK kinaseinhibitor. In other embodiments, said protein kinase inhibitor is a PLK1kinase inhibitor.

This invention may also be used in methods other than those involvingadministration to a patient.

One aspect of the invention relates to inhibiting protein kinaseactivity in a biological sample or a patient, which method comprisescontacting said biological sample with a compound of formula or acomposition comprising said compound. The term “biological sample”, asused herein, means a sample that is not in vivo such an in vitro or anex vivo sample, including, without limitation, cell cultures or extractsthereof; biopsied material obtained from a mammal or extracts thereof;and blood, saliva, urine, feces, semen, tears, or other body fluids orextracts thereof.

Inhibition of protein kinase activity in a biological sample is usefulfor a variety of purposes that are known to one of skill in the art.Examples of such purposes include, but are not limited to, bloodtransfusion, organ-transplantation, and biological specimen storage.

Another aspect of this invention relates to the study of protein kinasesin biological and pathological phenomena; the study of intracellularsignal transduction pathways mediated by such protein kinases; and thecomparative evaluation of new protein kinase inhibitors. Examples ofsuch uses include, but are not limited to, biological assays such asenzyme assays and cell-based assays.

The compounds of this invention may be prepared in general by methodsknown to those skilled in the art. Those compounds may be analyzed byknown methods, including but not limited to LCMS (liquid chromatographymass spectrometry) and NMR (nuclear magnetic resonance). Compounds ofthis invention may be also tested according to these examples. It shouldbe understood that the specific conditions shown below are onlyexamples, and are not meant to limit the scope of the conditions thatcan be used for making, analyzing, or testing the compounds of thisinvention. Instead, this invention also includes conditions known tothose skilled in that art for making, analyzing, and testing thecompounds of this invention.,

EXAMPLES

As used herein, the term “Rt(min)” refers to the HPLC retention time, inminutes, associated with the compound. Unless otherwise indicated, theHPLC method utilized to obtain the reported retention time is asfollows:

-   -   Column: ACE C8 column, 4.6×150 mm    -   Gradient: 0-100% acetonitrile+methanol 60:40 (20 mM Tris        phosphate)    -   Flow rate: 1.5 mL/minute    -   Detection: 225 nm.

Mass spec. samples were analyzed on a MicroMass Quattro Micro massspectrometer operated in single MS mode with electrospray ionization.Samples were introduced into the mass spectrometer using chromatography.

¹H-NMR spectra were recorded at 400 MHz using a Bruker DPX 400instrument. The following compounds of formula I were prepared andanalyzed as follows.

Example 13-[1-(2-Chloro-phenyl)-ethoxy]-5-(3-phenylamino-[1,2,4]triazol-1-yl)-thiophene-2-carboxylicacid amide (I-1)

Method A:3-Hydroxy-5-(3-phenylamino-[1,2,4]triazol-1-yl)-thiophene-2-carboxylicacid methyl ester

Phenyl-(1H-[1,2,4]triazol-3-yl)-amine (646 mg, 4.06 mmol, 1.15 Eq.) and2-Chloro-3-oxo-2,3-dihydro-thiophene-2-carboxylic acid methyl ester (680mg, 3.53 mmol, 1.0 Eq.) were dissolved in anhydrous MeCN (10 mL) and DMF(3 mL) and heated at reflux for 18 hours. After cooling to ambienttemperature, the resultant precipitate was isolated by filtration andwashed with DCM to give the sub-title compound as a brown solid (415 mg,1.31 mmol 37%); ¹H NMR (400 MHz, CDCl₃) δ 3.77 (3H, s), 6.89 (1H, t),7.11 (1H, s), 7.31 (2H, t), 7.57 (2H, t), 9.15 (1H, s), 9.70 (1H, s),10.78 (1H, s).

Method B:3-[1-(2-Chloro-phenyl)-ethoxy]-5-(3-phenylamino-[1,2,4]triazol-1-yl)-thiophene-2-carboxylicacid methyl ester

Triphenyl phosphine (124 mg, 0.47 mmol, 1.5 Eq.) was added to a stirredsolution of 1-(2-Chloro-phenyl)-ethanol (74 mg, 0.47 mmol, 1.5 Eq.) inanhydrous THF (5 mL) at 0° C. under nitrogen. The reaction was stirredat this temperature for 30 minutes, then diethyl azodicarboxylate (75μL, 0.47 mmol, 1.5 Eq.) was added-dropwise and the reaction stirred at0° C. for a further 1 hour.3-Hydroxy-5-(3-phenylamino-[1,2,4]triazol-1-yl)-thiophene-2-carboxylicacid methyl-ester (100 mg, 0.32 mmol, 1.0 Eq.) in anhydrous DMF (2 mL)was added and the reaction stirred for a further 1 hour at 0° C. thenallowed to warm to ambient temperature overnight. The crude reactionmixture was partitioned between EtOAc (50 mL) and water (50 mL). Theorganic phase was washed with water (2×20 mL), dried (MgSO₄) andconcentrated in vacuo. The crude product was purified by columnchromatography (ISCO Companion™, 12 g column, 0-100% EtOAc/petroleumEther) to give the sub-title compound as an off-white solid (88 mg, 0.19mmol, 73%); ¹H NMR (400 MHz, CDCl₃) δ 1.74 (3H, d), 3.93 (3H, s), 5.84(1H, q), 6.70 (1 h, br s), 6.74 (1H, s), 7.02 (1H, t), 7.21-7.40 (5H,m), 7.51 (1H, d), 7.63 (1H, d), 8.12 (1H, s).

Method C:3-[1-(2-Chloro-phenyl)-ethoxy]-5-(3-phenylamino-[1,2,4]triazol-1-yl)-thiophene-2-carboxylicacid amide (I-1)

3-[1-(2-Chloro-phenyl)-ethoxy]-5-(3-phenylamino-[1,2,4]triazol-1-yl)-thiophene-2-carboxylicacid methyl ester (66 mg, 0.15 mmol, 1.0 Eq.) was suspended in 7M NH₃ inMeOH (10 mL) and heated to 100° C. in a pressure tube for 3 days. Thesolvent was removed in vacuo and the product isolated by columnchromatography (10% MeOH/DCM) followed by tritruation from diethyl etherto give the title compound as an off-white solid (17 mg, 0.04 mmol,27%); ¹H NMR (400 MHz, d-6 DMSO) δ_(.) 1.72 (3H, d), 5.88 (1H, q), 6.88(1H, t), 7.03 (1H, br s), 7.26 (1H, s), 7.30 (2H, t), 7.36-7.43 (2H, m),7.52 (1H, dd), 7.57 (2H, d), 7.66 (1H, dd), 7.75 (1H, br s), 9.02 (1H,br s), 9.65 (1H, s).

Example 23-[1-(2-Chloro-phenyl)-ethoxy]-5-[3-(2,2-difluoro-benzo[1,3]dioxol-4-ylamino)-[1,2,4]triazol-1-yl]-thiophene-2-carboxylicacid amide (I-2)

Method D:(2,2-Difluoro-benzo[1,3]dioxol-4-yl)-(1H-[1,2,4]triazol-3-yl)-amine

2,2-Difluoro-benzo[1,3]dioxol-4-yl-cyanamide (1.0 g, 5.05 mmol, 1.0 Eq.)was dissolved in MeOH (50 mL) and water (10 mL). Hydrazine hydrate (245μL, 5.05 mmol, 1.0 Eq.) was added followed by 32 wt % aqueousformaldehyde (474 mL, 5.05 mmol, 1.0 Eq.) and the reaction stirred atambient temperature overnight. The solvent was removed in vacuo, and theresidue recrystalised from MeOH to give the sub-title compound as anoff-white solid (516 mg, 2.15 mmol, 42%); ¹H NMR (400 MHz, d-6 DMSO)δ_(.) 6.88 (1H, d), 7.10 (1H, t), 7.72 (1H, d), 8.09 (1H, br s), 9.25(1H, br s), 13.20 (1H, br s); ¹⁹F NMR (376 MHz, d-6 DMSO, protondecoupled) δ −49.1.

This intermediate was involved in the sequence described in Methods A-Cto give the title compound as an off-white solid (19 mg, 0.04 mmol,19%); ¹H NMR (400 MHz, d-6 DMSO) δ_(.) 1.72 (3H, d), 5.87 (1H, q), 7.00(2H, d+br s), 7.18 (1H, t), 7.29 (1H, s), 7.32-7.42 (2H, m), 7.51 (1H,dd), 7.64-7.68 (2H, m), 7.93 (1H, br s), 9.04 (1H, s), 9.75 (1H, s); ¹⁹FNMR (376 MHz, d-6 DMSO, proton decoupled) δ −40.1.

Example 35-[3-(3-Chloro-phenylamino)-[1,2,4]triazol-1-yl]-3-[1-(2-chloro-phenyl)-ethoxy]-thiophene-2-carboxylicacid amide (I-3)

Prepared from 3-Chloro-phenyl-cyanamide using Methods A-D above andpurified by tritruation from EtoAc to give the title compound a paleyellow solid (5 mg, 0.01 mmol, 5%); ¹H NMR (400 MHz, d-6 DMSO) δ 1.72(3H, s), 5.89 (1H, q), 6.92 (1H, dd), 7.04 (1H, br s), 7.27 (1H, s),7.29-7.53 (5H, m), 7.65-7.70 (2H, m), 7.70 (1 h, br s), 9.05 (1H, s),9.90 (1H, s).

HPLC # MS+ ¹HNMR Rt/min I-1 440.35 ¹HNMR (DMSO) 1.72 (3H, d), 5.88 (1H,q), 9.65 6.88 (1H, t), 7.03 (1H, br s), 7.25-7.31 (3H, m), 7.36-7.43(2H, m), 7.52 (1H, d), 7.57 (2H, d), 7.66 (1H, dd), 7.75 (1H, br s),9.02 (1H, br s), 9.65 (1H, s); I-2 520.70 ¹HMMR (DMSO) 1.72 (3H, d),5.87 (1H, q), 10.4 7.00 (2H, d + br s), 7.18 (1H, t), 7.29 (1H, s),7.32-7.42 (2H, m), 7.51 (1H, dd), 7.64-7.68 (2H, m), 7.93 (1H, br s),9.04 (1H, s), 9.75 (1H, s) I-3 474.70 ¹HNMR (DMSO) 1.72 (3H, s), 5.89(1H, q), 10.3 6.92 (1H, dd), 7.04 (1H, br s), 7.27 (1H, s), 7.29-7.53(5H, m), 7.65-7.70 (2H, m), 7.70 (1h, br s), 9.05 (1H, s), 9.90 (1H, s)

Example 4 PLK Assays

The compounds of the present invention are evaluated as inhibitors ofhuman PLK kinase using the following assays.

Plk1 Inhibition Assay

Compounds were screened for their ability to inhibit Plk1 using aradioactive-phosphate incorporation assay.

Assays were carried out in a mixture of 25 mM HEPES (pH 7.5), 10 mMMgCl₂, and 1 mM DTT. Final substrate concentrations were 50 μM[γ-33P]ATP (136 mCi 33P ATP/mmol ATP, Amersham Pharmacia Biotech/SigmaChemicals) and 10 μM peptide (SAM68 protein Δ332-443). Assays werecarried out at 25° C. in the presence of 15 nM Plk1 (A20-K338). An assaystock buffer solution was prepared containing all of the reagents listedabove, with the exception of ATP and the test compound of interest. 30μL of the stock solution was placed in a 96 well plate followed byaddition of 2 μL of DMSO stock containing serial dilutions of the testcompound (typically starting from a final concentration of 10 μM with2-fold serial dilutions) in duplicate (final DMSO concentration 5%). Theplate was pre-incubated for 10 minutes at 25° C. and the reactioninitiated by addition of 8 μL [γ-33P]ATP (final concentration 50 μM).

The reaction was stopped after 60 minutes by the addition of 100 μL0.14M phosphoric acid. A multiscreen phosphocellulose filter 96-wellplate (Millipore, Cat no. MAPHN0B50) was pretreated with 100 μL 0.2Mphosphoric acid prior to the addition of 125 μL of the stopped assaymixture. The plate was washed with 4×200 μL 0.2M phosphoric acid. Afterdrying, 10 μL Optiphase ‘SuperMix’ liquid scintillation cocktail (PerkinElmer) was added to the well prior to scintillation counting (1450Microbeta Liquid Scintillation Counter, Wallac).

After removing mean background values for all of the data points,Ki(app) data were calculated from non-linear regression analysis of theinitial rate data using the Prism software package (GraphPad Prismversion 3.0cx for Macintosh, GraphPad Software, San Diego Calif., USA).

Plk1 Inhibition Assay

Compounds were screened for their ability to inhibit Plk1 using aradioactive-phosphate incorporation assay. Assays were carried out in amixture of 25 mM HEPES (pH 7.5), 10 mM MgCl₂, 0.1% BSA, and 2 mM DTT.Final substrate concentrations were 150 μM [γ-33P]ATP (115 mCi 33PATP/mmol ATP, Amersham Pharmacia Biotech/Sigma Chemicals) and 300 μMpeptide (KKKISDELMDATFADQEAK (SEQ ID NO: 1)). Assays were carried out at25° C. in the presence of 4 nM Plk1. An assay stock buffer solution wasprepared containing all of the reagents listed above, with the exceptionof ATP and the test compound of interest. 30 μL of the stock solutionwas placed in a 96 well plate followed by addition of 2 μL of DMSO stockcontaining serial dilutions of the test compound (typically startingfrom a final concentration of 10 μM with 2-fold serial dilutions) induplicate (final DMSO concentration 5%). The plate was pre-incubated for10 minutes at 25° C. and the reaction initiated by addition of 8 μL[γ-33P]ATP (final concentration 150 μM).

The reaction was stopped after 90 minutes by the addition of 100 μL0.14M phosphoric acid. A multiscreen phosphocellulose filter 96-wellplate (Millipore, Cat no. MAPHN0B50) was pretreated with 100 μL 0.2Mphosphoric acid prior to the addition of 125 μL of the stopped assaymixture. The plate was washed with 4×200 μL 0.2M phosphoric acid. Afterdrying, 100 μL Optiphase ‘SuperMix’ liquid scintillation cocktail(Perkin Elmer) was added to the well prior to scintillation counting(1450 Microbeta Liquid Scintillation Counter, Wallac).

After removing mean background values for all of the data points,Ki(app) data were calculated from non-linear regression analysis of theinitial rate data using the Prism software package (GraphPad Prismversion 3.0cx for Macintosh, GraphPad Software, San Diego Calif., USA).

In general, compounds of the invention are effective for the inhibitionof Plk1. The following compounds showed Ki between 10 nM and 100 nM inthe radioactive incorporation assay: I-1, I-2, and I-3.

Plk2 Inhibition Assay

Compounds were screened for their ability to inhibit Plk2 using aradioactive-phosphate incorporation assay. Assays were carried out in amixture of 25 mM HEPES (pH 7.5), 10 mM MgCl₂, 0.1% BSA, and 2 mM DTT.Final substrate concentrations were 200 μM [γ-33P]ATP (57 mCi 33PATP/mmol ATP, Amersham Pharmacia Biotech/Sigma Chemicals) and 300 μMpeptide (KKKISDELMDATFADQEAK (SEQ ID NO: 1)). Assays were carried out at25° C. in the presence of 25 nM Plk2. An assay stock buffer solution wasprepared containing all of the reagents listed above, with the exceptionof ATP and the test compound of interest. 30 μL of the stock solutionwas placed in a 96 well plate followed by addition of 2 μL of DMSO stockcontaining serial dilutions of the test compound (typically startingfrom a final concentration of 10 μM with 2-fold serial dilutions) induplicate (final DMSO concentration 5%). The plate was pre-incubated for10 minutes at 25° C. and the reaction initiated by addition of 8 μL[γ-33P]ATP (final concentration 200 μM).

The reaction was stopped after 90 minutes by the addition of 10 μL 0.14Mphosphoric acid. A multiscreen phosphocellulose filter 96-well plate(Millipore, Cat no. MAPHN0B50) was pretreated with 100 μL 0.2Mphosphoric acid prior to the addition of 125 μL of the stopped assaymixture. The plate was washed with 4×200 μL 0.2M phosphoric acid. Afterdrying, 100 μL Optiphase ‘SuperMix’ liquid scintillation cocktail(Perkin Elmer) was added to the well prior to scintillation counting(1450 Microbeta Liquid Scintillation Counter, Wallac).

After removing mean background values for all of the data points,Ki(app) data were calculated from non-linear regression analysis of theinitial rate data using the Prism software package (GraphPad Prismversion 3.0 cx for Macintosh, GraphPad Software, San Diego Calif., USA).

Plk3 Inhibition Assay

Compounds were screened for their ability to inhibit Plk3 using aradioactive-phosphate incorporation assay. Assays were carried out in amixture of 25 mM HEPES (pH 7.5), 10 mM MgCl₂, and 1 mM DTT. Finalsubstrate concentrations were 75 μM [γ-33P]ATP (60 mCi 33P ATP/mmol ATP,Amersham Pharmacia Biotech/Sigma Chemicals) and 10 μM peptide (SAM68protein Δ332-443). Assays were carried out at 25° C. in the presence of5 nM Plk3 (S38-A340). An assay stock buffer solution was preparedcontaining all of the reagents listed above, with the exception of ATPand the test compound of interest. 30 μL of the stock solution wasplaced in a 96 well plate followed by addition of 2 μL of DMSO stockcontaining serial dilutions of the test compound (typically startingfrom a final concentration of 10 μM with 2-fold serial dilutions) induplicate (final DMSO concentration 5%). The plate was pre-incubated for10 minutes at 25° C. and the reaction initiated by addition of 8 μL[γ-33P]ATP (final concentration 75 μM).

The reaction was stopped after 60 minutes by the addition of 100 μL0.14M phosphoric acid. A multiscreen phosphocellulose filter 96-wellplate (Millipore, Cat no. MAPHN0B50) was pretreated with 100 μL 0.2Mphosphoric acid prior to the addition of 125 μL of the stopped assaymixture. The plate was washed with 4×200 μL 0.2M phosphoric acid. Afterdrying, 100 μL Optiphase ‘SuperMix’ liquid scintillation cocktail(Perkin Elmer) was added to the well prior to scintillation counting(1450 Microbeta Liquid Scintillation Counter, Wallac).

After removing mean background values for all of the data points,Ki(app) data were calculated from non-linear regression analysis of theinitial rate data using the Prism software package (GraphPad Prismversion 3.0cx for Macintosh, GraphPad Software, San Diego Calif., USA).

Plk4 Inhibition Assay

Compounds are screened for their ability to inhibit Plk4 using aradioactive-phosphate incorporation assay. Assays are carried out in amixture of 8 mM MOPS (pH 7.5), 10 mM MgCl2, 0.1% BSA and 2 mM DTT. Finalsubstrate concentrations are 15 μM [γ-33P]ATP (227 mCi 33P ATP/mmol ATP,Amersham Pharmacia Biotech/Sigma Chemicals) and 300 μM peptide(KKKMDATFADQ (SEQ ID NO: 2)). Assays are carried out at 25° C. in thepresence of 25 nM Plk4. An assay stock buffer solution is preparedcontaining all of the reagents listed above, with the exception of ATPand the test compound of interest. 30 μL of the stock solution is placedin a 96 well plate followed by addition of 2 μL of DMSO stock containingserial dilutions of the test compound (typically starting from a finalconcentration of 10 μM with 2-fold serial dilutions) in duplicate (finalDMSO concentration 5%). The plate is pre-incubated for 10 minutes at 25°C. and the reaction initiated by addition of 8 μL [γ-33P]ATP (finalconcentration 15 μM).

The reaction is stopped after 180 minutes by the addition of 100 μL0.14M phosphoric acid. A multiscreen phosphocellulose filter 96-wellplate (Millipore, Cat no. MAPHN0B50) is pretreated with 100 μL 0.2Mphosphoric acid prior to the addition of 125 μL of the stopped assaymixture. The plate is washed with 4×200 μL 0.2M phosphoric acid. Afterdrying, 100 μL Optiphase ‘SuperMix’ liquid scintillation cocktail(Perkin Elmer) is added to the well prior to scintillation counting(1450 Microbeta Liquid Scintillation Counter, Wallac).

After removing mean background values for all of the data points,Ki(app) data are calculated from non-linear regression analysis of theinitial rate data using the Prism software package (GraphPad Prismversion 3.0cx for Macintosh, GraphPad Software, San Diego Calif., USA).

While we have described a number of embodiments of this invention, it isapparent that our basic examples may be altered to provide otherembodiments that utilize the compounds, methods, and processes of thisinvention. Therefore, it will be appreciated that the scope of thisinvention is to be defined by the appended claims rather than by thespecific embodiments that have been represented by way of exampleherein.

1. A compound of formula I:

wherein R¹ is H, C₁₋₆aliphatic, or C₃₋₆cycloaliphatic; G is —C(R)₂— or—O—; L is C₀₋₃aliphatic optionally substituted with 0-3 J^(L); Ring A istriazolyl optionally substituted with 0-3 J^(A); Ring B is 5-6 memberedaromatic monocyclic ring containing 0-3 heteroatoms selected from O, N,and S; Ring B is optionally substituted with 0-5 J^(B) and optionallyfused to Ring B′; Ring B′ is a 5-8 membered aromatic or nonaromaticmonocyclic ring containing 0-3 heteroatoms selected from O, N, and S;Ring B′ is optionally substituted with 0-4 J^(B′); each J^(A), J^(B),and J^(B′) is independently C₁₋₆haloalkyl, halo, NO₂, CN, Q, or —Z-Q; Zis independently C₁₋₆aliphatic optionally interrupted with 0-3occurrences of —NR—, —O—, —S—, —C(O)—, —C(═NR)—, —C(═NOR)—, —SO—, or—SO₂—; each Z is optionally substituted with 0-2 J^(Z); Q is H; C₁₋₆aliphatic; a 3-8-membered aromatic or nonaromatic monocyclic ring having0-3 heteroatoms independently selected from O, N, and S; or an 8-12membered aromatic or nonaromatic bicyclic ring system having 0-5heteroatoms independently selected from O, N, and S; each Q isoptionally substituted with 0-5 J^(Q); each J^(L) and J^(Z) isindependently H, halo, C₁₋₆ aliphatic, C₃₋₆cycloaliphatic, NO₂, CN,—NH₂, —NH(C₁₋₄ aliphatic), —N(C₁₋₄ aliphatic)₂, —OH, —O(C₁₋₄ aliphatic),—CO₂H, —CO₂(C₁₋₄ aliphatic), —O(haloC₁₋₄ aliphatic), or halo(C₁₋₄aliphatic); each J^(Q) is independently M or —Y-M; each Y isindependently an unsubstituted C₁₋₆aliphatic optionally interrupted with0-3 occurrences of —NR—, —O—, —S—, —C(O)—, —SO—, or —SO₂—; each M isindependently H, C₁₋₆ aliphatic, C₃₋₆cycloaliphatic, halo(C₁₋₄aliphatic), —O(haloC₁₋₄ aliphatic), C₃₋₆ heterocyclyl, halo, NO₂, CN,OH, OR′, SH, SR′, NH₂, NHR′, N(R′)₂, COH, COR′, CONH₂, CONHR′, CONR′₂,NHCOR′, NR′COR′, NHCONH₂, NHCONHR′, NHCON(R′)₂, SO₂NH₂, SO₂NHR′,SO₂N(R′)₂, NHSO₂R′, or NR′SO₂R′; R is H or unsubstituted C₁₋₆aliphatic;R′ is unsubstituted C₁₋₆aliphatic; or two R′ groups, together with theatom to which they are bound, form an unsubstituted 3-8 memberednonaromatic monocyclic ring having 0-1 heteroatoms independentlyselected from O, N, and S.
 2. The compound of claim 1, wherein G is—C(R)₂—.
 3. The compound of claim 1, wherein G is O.
 4. The compound ofclaim 3, wherein R¹ is H.
 5. The compound of claim 4, wherein Ring A is


6. The compound of claim 5, wherein Ring A is


7. The compound of claim 5, wherein Ring B is a 6 membered aromatic ringcontaining 0-2 nitrogen atoms and Ring B is optionally substituted with0-5 J^(B).
 8. The compound of claim 7, wherein Ring B is fused to RingB′ and Ring B is optionally substituted with 0-5 J^(B) and Ring B′ isoptionally substituted with 0-4 J^(B′).
 9. The compound of claim 8,wherein Ring B′ is a 5-6 membered aromatic ring containing 0-3heteroatoms selected from O, N, and S, wherein Ring B′ is optionallysubstituted with 0-4 J^(B′).
 10. The compound of claim 5, wherein J^(A)is H, C₁₋₆ aliphatic, C₃₋₆cycloaliphatic, halo(C₁₋₄ aliphatic),—O(haloC₁₋₄ aliphatic), C₃₋₆ heterocyclyl, halo, NO₂, CN, OH, OR, SH,SR, NH₂, NHR, N(R)₂, COH, COR, CONH₂, CONHR, CONR₂, NHCOR, NRCOR,NHCONH₂, NHCONHR, NHCON(R)₂, SO₂NH₂, SO₂NHR, SO₂N(R)₂, NHSO₂R, orNRSO₂R.
 11. The compound of claim 10, wherein J^(A) is H.
 12. Thecompound of claim 7, wherein J^(B) is H, C₁₋₆ aliphatic,C₃₋₆cycloaliphatic, halo(C₁₋₄ aliphatic), —O(haloC₁₋₄ aliphatic), C₃₋₆heterocyclyl, halo, NO₂, CN, OH, OR, SH, SR, NH₂, NHR, N(R)₂, COH, COR,CONH₂, CONHR, CONR₂, NHCOR, NRCOR, NHCONH₂, NHCONHR, NHCON(R)₂, SO₂NH₂,SO₂NHR, SO₂N(R)₂, NHSO₂R, or NRSO₂R.
 13. The compound of claim 9,wherein J^(B′) is H, C₁₋₆ aliphatic, C₃₋₆cycloaliphatic, halo(C₁₋₄aliphatic), —O(haloC₁₋₄ aliphatic), C₃₋₆ heterocyclyl, halo, NO₂, CN,OH, OR, SH, SR, NH₂, NHR, N(R)₂, COH, COR, CONH₂, CONHR, CONR₂, NHCOR,NRCOR, NHCONH₂, NHCONHR, NHCON(R)₂, SO₂NH₂, SO₂NHR, SO₂N(R)₂, NHSO₂R, orNRSO₂R.
 14. A compound selected from the group consisting of:


15. A composition comprising a compound of claim 1, and apharmaceutically acceptable carrier, adjuvant, or vehicle.
 16. A methodof inhibiting protein kinase activity in a biological sample comprisingcontacting said biological sample with: a compound of claim 1, whereinsaid protein kinase is PLK1.
 17. A method of treating melanoma, myeloma,leukemia, lymphoma, neuroblastoma, or a cancer selected from colon,breast, gastric, ovarian, cervical, lung, central nervous system (CNS),renal, prostate, bladder, or pancreatic, in a patient wherein saidmethod comprises administering to said patient a compound of claim 1.18. A method of treating cancer in a patient wherein said methodcomprises administering to said patient a compound of claim
 1. 19. Aprocess for preparing a compound of formula I:

wherein G is O, R¹ is H, and Ring A, Ring B, J^(A), J^(B) and L are asdefined according to claim 1, comprising reacting a compound of formula3

wherein G is O and Ring A, Ring B, J^(A), J^(B) and L are as definedaccording to claim 1, under suitable amide forming conditions to formthe compound of formula I, wherein the suitable amide forming conditionsinclude heating the compound of formula 3 in the presence of NH₃/MeOH.20. A composition comprising a compound of claim 14, and apharmaceutically acceptable carrier, adjuvant, or vehicle.
 21. A methodof inhibiting protein kinase activity in a biological sample comprisingcontacting said biological sample with a compound of claim 14, whereinsaid protein kinase is PLK1.
 22. A method of treating melanoma, myeloma,leukemia, lymphoma, neuroblastoma, or a cancer selected from colon,breast, gastric, ovarian, cervical, lung, central nervous system (CNS),renal, prostate, bladder, or pancreatic, in a patient, wherein saidmethod comprises administering to said patient a compound of claim 14.23. A method of treating cancer in a patient, wherein said methodcomprises administering to said patient a compound of claim 14.